Coxiella burnetii, a Gram-negative obligate intracellular bacterium, is the etiological agent of acute and chronic Q (query) fever in humans. Analysis of C. burnetii virulence genes has been hampered by the inability to generate and isolate specific mutations and relies to date on the characterization of gene expression in heterologous hosts. Introduction of stably maintained exogenous plasmid DNA was first reported over 10 years ago, but subsequent reports and unreported attempts to utilize this shuttle vector were disappointing. Recent advances in the field such as, extracellular growth, defined selection markers and a shuttle vector start to overcome these difficulties and allow adaptation and refinement of existent classical genetic methods to this specific microorganism. The objective of this project is to develop targeted gene disruption in C. burnetii using allelic exchange or the retrohoming mechanism of the mobile group II intron LtrB of Lactobacillus lactis (TargeTron technology, Sigma-Aldrich). We will accomplish the objective by pursuing the following specific aims: (1) Development of a shuttle vector for controlled gene expression in C. burnetii. In C. burnetii homologous recombination is mediated by the AddAB complex, which is associated with a low recombination frequency. To overcome this hurdle we will establish controlled gene expression by testing reporter gene expression from widely used inducible promoter in C. burnetii. This will support the introduction of heterologous recombination systems in C. burnetii and usage of counter selection markers to force allelic exchange. (2) Development of site-specific mutagenesis in C. burnetii using allelic exchange. We will achieve allelic exchange in C. burnetii based on the developed Coxiella-specific shuttle vector. Inactivated genes will be delivered on the stable replicating Coxiella vector with a curable, for replication temperature-sensitive origin. This approach provides a higher amount of substrate DNA, when compared to classical suicide vectors, prior to the forced integration event and might lead to a higher success rate. (3) Development of site-specific mutagenesis in C. burnetii using the TargeTron system. We will achieve targeted gene disruption by adapting the TargeTron Technology, based on the re-programmable mobile group II intron of L. lactis for C. burnetii. In all three approaches we will specifically target genes, which inactivation results in a screenable phenotype; Mutation of rpsL results in streptomycin resistance and inactivation of waaF to LPS core truncation or com1 or sodC as immunogenic proteins as alternatives. Development of targeted gene disruption would be novel for C. burnetii and a major contribution to new approaches to investigate the pathogenic process of Q fever. PUBLIC HEALTH RELEVANCE: Infections of humans with Coxiella burnetii, a potential bioterrorism agent, manifest as acute, flu-like illness or as chronic inflammation of the heart or liver. The lack of genetic tools for this microorganism has hampered the identification of factors which allow the organism to infect and persist in humans. The proposed project outlined several strategies to develop genetic tools for characterization of gene functions and will aid the development of a vaccine as national security goal.